3 CHRONIC KIDNEY DISEASES (CKDs) [1]:Definition: Any condition that causes reduced kidney function over a period of time is a chronic kidney disease.CKD is present when a patient’s glomerular filtration rate remains below 60 mL min-1 for more than 3 months, or when a patient’s urine albumin-to-creatinine ratio is over 30 mg of albumin for each g of creatinine (30 mg g-1).Glomerular filtration rateGlomerular filtration rate (GFR) is a test used to check how well the kidneys are working. Specifically, it estimates how much blood passes through the glomeruli each minute. Glomeruli are the tiny filters in the kidneys that filter waste from the blood (MEDLINE PLUS).Albumin is a protein found in the blood. A healthy kidney does not let albumin pass into the urine. A damaged kidney lets some albumin pass into the urine. The less albumin in your urine, the better. (National Kidney Disease Education Program of USA.)1. Kidney Disease Statistics for the United States, National Kidney and Urologic Diseases Information Clearinghouse (NKUDIC), Accessed on July 5th, 2014.

4 SKIPCreatinine is removed from the blood chiefly by the kidneys, primarily by glomerular filtration, but also by proximal tubular secretion.Little or no tubular reabsorption of creatinine occurs.If the filtration in the kidney is deficient, creatinine blood levels rise.Therefore, creatinine levels in blood and urine may be used to calculate the creatinine clearance (CrCl), which correlates with the glomerular filtration rate (GFR).Blood creatinine levels may also be used alone to calculate the estimated GFR (eGFR).

5 CHRONIC KIDNEY DISEASES (CKDs) ARE NOT UNCOMMON [1-5]One in 10 American adults, more than 20 million, have some level of CKD [1-3] due to diabetes or hypertension.Around 1.7 million Australians (1 in 10) aged ≥ 18 years have clinical evidence of CKD [4,5] due to diabetes or hypertension .[1] Kidney Disease Statistics for the United States, National Kidney and Urologic Diseases Information Clearinghouse (NKUDIC),http://kidney.niddk.nih.gov/ kudiseases /pubs /kustats/#16.[2] National Kidney and Urologic Diseases Information Clearing House (NKUDIC), U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES.[3] Centers for Disease Control and Prevention, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES , NIH Publication No. 12–3895, November 15, 2012.[4] STATE OF THE NATION: CHRONIC KIDNEY DISEASE IN AUSTRALIA, MAY 2014, KIDNEY HEALTH AUSTRALIA: Accessed 03rd July, 2014.[5] ANZDATA, Summary of Australian and New Zealand Dialysis and Transplantation Registry, South Australia, Adlaide, 2013.

6 CKDu IN SRI LANKA CKDu: Chronic Kidney Disease of unknown AetiologyAffected AreasGirandurukotte in the Badulla DistrictMedawachchiya and Padaviya in the Anuradhapura DistrictMedirigiriya and Hingurakgoda in the Polonnaruwa DistrictNickawewa in the Kurunagala District

8 FACTS ABOUT CKDu IN SRI LANKADiscovered since mid 1990’s.Affected patients are mostly rice paddy farmers.The estimated age-standardized prevalence of the disease is 15% [7].According to the Ministry of Health, the estimated number of CKDu cases is 69,258 as of June 2014 [8, 9].Estimated death toll is 20,000 [8].Affected patients were of age categories over 55 years [10].The age category is shifting towards younger ages [10].It has been predicted that even the school children could be affected in the near future [10].[7] N. Jayathilaka, S. Mendis, P. Maheepala and F.A. Metha, “Chronic Kidney Disease of Unknown Aeitiology: Prevalence and Causative Factors in a Developing Country”, BMC Nephrology, 14 (2013) doi: /[8] Channa Jayasumana, Sarath Gunathilaka and Priyantha Senanayake, “Glyphosate, Hard Water, and Nephrotoxic Metals: Are They the Culprit Behind the Epidemic of Chronic Kidney Disease of Unknown Etiology in Sri Lnka?”, International Journal of Environmental Research and Public Health, 11 (2014)[9] Private Communication with Dr. Channa Jayasumana[10] Channa Jayasumana, Lakbima, Sunday Edition, 22nd June, 2014.

9 UNIQUE FEATURES OF CKDuIt is said that CKDu aetiology does not include commonly known risk factors for a chronic kidney disease.No Diabetes mellitusNo HypertensionNo Glomerulonephritis [8, 11]SO, IT IS A VERY SPECIAL DISEASE!!!Glomerulonephritis, also known as glomerular nephritis, is a term used to refer to several renal diseases (usually affecting both kidneys). Many of the diseases are characterised by inflammation either of the glomeruli or small blood vessels in the kidneys, hence the name.[8] Channa Jayasumana, Sarath Gunathilaka and Priyantha Senanayake, “Glyphosate, Hard Water, and Nephrotoxic Metals: Are They the Culprit Behind the Epidemic of Chronic Kidney Disease of Unknown Etiology in Sri Lnka?”, International Journal of Environmental Research and Public Health, 11 (2014)[11] N.T. Athuraliya, T.D. Abeysekara, P.H. Amerasinghe, R. Kumarasiri, P. Bandara, U. Karunaratna, A.H. Milton and A. L. Jones, “Uncertain etiologies of Proteinuric Chronic Kidney Disease in Rural Sri Lanka”, Kidney International, 80 (2011)

12 IMPORTANT POINTS TO CONSIDERPatients are confined to a unique geographical area of the country.Disease was not found about 25 years ago.Number of patients is continuously increasing very rapidly now.Affected people are mostly rice paddy farmers.Patients are mainly males.The disease is prevalent in the region where fluorosis is also prevalent.In general, drinking water has high salinity.

13 IMPLICATIONS!The aeitiology of the disease may be associated with local environmental and occupational conditions, food habits or may be due to genetic reasons .Occupation of the patients is already known. They are mostly rice paddy farmers.Look for unique environmental conditions of the region and consequences of farming.Farming communities in NCP and EP of Sri Lanka comprise of native people of the region plus those migrated from other areas, such as, Gampaha, Kurunegala, Kegalle, Galle, Matara, Hambantota, Kotmale etc. during the colonization period. Better to see whether there are any Genetic or Hereditary Factors such as the Patients are of Particular Tribe Living in this Area and so on.Better to investigate their food habits, habits such as chewing “BULATHWITA”. Consumption of illicit alcohols and inadequate water consumption during hard work of farming. Do they smoke? Smoking is the biggest source of cadmium intake.

14 GEOCHEMICAL ASPECTS-CHRONIC RENAL FAILURE [15]First Study by C.B. Dissanayake in 2005.Dental fluorosis and Kidney Diseases are commonly observed in Sri Lanka in the same geographical area.Chronic Renal Failure has been observed in the Areas of Anuradhapura and Polonnaruwa Districts.Geochemically, water in these areas are Na/K type with Cl- subtype predominating due to increasing drought conditions and consequent salt building up (This means water hardness is mostly due to permanent hardness and most of the compounds of sodium and potassium are water soluble!).The Total Dissolved Solids and Electrical Conductivity are high.Unique feature is that F- in ground waters of some areas is 10 mg L-1 which is much higher than maximum tolerable F- concentration in drinking water of 1.5 mg L-1 which has now reduced down to 0.5 mg L-1 (WHO Limit, vide infra).[15] C.B. Dissnayake, “Water Quality in the Dry Zone of Sri Lanka-Some Interesting Health Aspects”, J. National Science Foundation of Sri Lanka, 53 (2005)

18 WORLD MAP WITH RESPECT TO FLUORIDE IN GROUND WATER SKIPMAP WAS PREPAED BY Mr. CHARITH ANURUDDHA, ONE OF MY RAs, BASED ON DATA GIVEN IN [19] .

19 Fluoride in Australian Waters [20]SKIPFluoride in Australian Waters [20]Very Low.Fluoridization of drinking water is required to prevent dental caries among small and teenage children.The recommendations for fluoride levels in Australian water fluoridation programs should comply with those set out in the National Health and Medical Research Council’s public statement .The efficacy and safety of fluoridation is 0.5 ppm.[20] “Fluoride concentration in Australia’s Drinking Waters”, Victoria, Department of Health, Accessed on 06/07/2014Accessed on

20 WHO STANDARDS OF FLUORIDE IN DRINKING-WATER: SKIPWHO noted that mottling of teeth (i.e. dental fluorosis) is sometimes associated with fluoride levels in drinking-water above 1.5 mg L–1 and crippling skeletal fluorosis can ensue when fluoride levels exceed 10 mg L–1.A guideline value of 1.5 mg L–1 was, therefore, recommended by WHO as a level at which dental fluorosis should be minimal (WHO, 1984).The 1.5 mg L–1 fluoride guideline value that was set in 1984 was subsequently re-evaluated by WHO and it was concluded that there was no evidence to suggest that it should be revised (WHO, 1996, 2004).The 1.5 mg L–1 guideline value of WHO is not a “fixed” value but is intended to be adapted to take account of local conditions (e.g. diet, water consumption, etc.).

21 FLUORIDE METABOLISM FLURIDE ABSORPTION BY THE HUMAN BODYApproximately 75–90% of ingested fluoride is absorbed.In an acidic stomach, fluoride is converted into hydrogen fluoride (HF) and up to about 40% of the ingested fluoride is absorbed from the stomach as HF (Discuss corrosiveness).Fluoride not absorbed in the stomach (60%) is absorbed in the intestine and is unaffected by pH at this site.Relative to the amount of fluoride ingested, high concentrations of cations that form insoluble complexes with fluoride (e.g. calcium, magnesium and aluminium) can markedly decrease gastrointestinal fluoride absorption.

22 FLUORIDE DISTRIBUTION IN THE HUMAN BODYOnce absorbed into the blood, fluoride is readily distributed throughout the body.Approximately 99% of the body burden of fluoride is retained in calcium rich areas such as bones and teeth (dentine and enamel).Fluoride is then incorporated into the hydroxyapatite [Ca10(PO4)6(OH)2] lattice through ion-exchange.Porous Ca10(PO4)6(OH)2(s) + xF-(blood) ⇌Denser Ca10(PO4)6(OH)2-xFx(s) + xOH-(blood)Out of eleven important functions of bones, detoxification is one of the most important functions.Toxic anions such as aresenite and arsenate in the blood are adsorbed by the hydroxyl groups of hydroxyapatite and thereby the supply to other organs is prevented.These anions are then slowly released to the kidney for safe discharge.When the OH groups are substituted with F- anions this mechanism is not possible.Ca2+ can be exchanged for Cd2+ in Cd2+ toxicity causing weak bones (osteoporosis) (vide infra)

23 STRUCTURE OF NATURAL BONENANOTECHNOLOGICAL ARCHITECTURE OF BONESCOMPACT AND SPONGY PARTSOF BONEA femur head (highest part of the thigh bone) with a cortex of compact bone and medulla of trabecular bone.

24 Tailor-made Prosthesis by the Bio-Chemical-Computer Science-Engineering- Medical Research Group of University of Peradeniya

25 EFFECTS OF FLUORIDE ON HUMANS SKIPA number of studies have reported on the acute effects of fluoride exposure following fluoridation overdosing.However, the effects of long-term exposure to naturally occurring fluoride from drinking-water and other environmental sources (Chronic effects) are the major concern with regard to human health.A large number of epidemiological studies have been conducted in many countries concerning the effects of long-term exposure to fluoride.

27 FACTS ON SKELETAL FLUOROSIS SKIPParaestheria: Sensation of tingling, tickling, prickling, pricking, or burning of a person's skin with no apparent long-term physical effect.Poker Back: Arthritis and osteitis deformans involving the spinal column.EXOSTOSIS: Formation of new bone on the surface of a bone.

28 MUTAGENECITY AND RELATED END-POINTS. SKIPStudies have been carried out with bacteria, insects, laboratory animals and in vitro studies with human cells.Number of positive results in a variety of mammalian cell types for chromosome damage (USNRC 1993, WHO 1996, IPCS 2000) have been observed.Effects are due to interferences with protein synthesis rather than by any direct interaction between F- and DNA.THIS IS TRUE SINCE F- IS EXTREMELY INTERT IN ITS’ F- FORM.

29 FLUORIDE AND CANCER SKIPMy Opinion: THE POSSIBILITY IS DUE TO VERY HIGH CORROSIVENESS OF HYDROGEN FLUORIDE SOLUTION. HF CAN ATTACK ANYTHING. Can Even Dissolve Glass! F- can precipitate certain important metal ions such as Ca2+ as CaF2(s). F- can attack enzymes inhibiting their catalytic functions. In the body, hydrofluoric acid reacts with the ubiquitous biologically important ions such as Ca2+ and Mg2+. Formation of insoluble calcium fluoride is proposed as the aetiology for both precipitous fall in serum calcium and the severe pain associated with tissue toxicity [21].[21] Hoffman, Robert S. et al. (2007) Goldfrank's Manual of Toxicologic Emergencies. New York: McGraw-Hill Professional, p. 1333, ISBN

31 TOXICITY OF FLUOROALUMINATESSKIPTOXICITY OF FLUOROALUMINATESSTUDIES WITH Nostoc linckia and Chlorella vulgarisBLUE-GREEN ALGAE

32 UPTAKE OF FLUOROALUMINATE SPECIES BY BLUE-GREEN ALGAE [23]SKIPUPTAKE OF FLUOROALUMINATE SPECIES BY BLUE-GREEN ALGAE [23]Have pH-dependent Mg2+ and Ca2+-ATPase (Class of enzymes that catalyze the decomposition of ATP into ADP and a free phosphate ion) activities.Toxicity increases with increasing acidity.Toxicity of fluoroaluminate is reduced in the presence of EDTA and citrate (THESE ARE BETTER CHEALATING AGENTS THAN AlF4-). AlF4− inhibits the ATPase activity by acting as a functional barrier without affecting the structure of the enzyme. This upsets the cell metabolism and the removal of toxins.[23] Yasmin Husaini, L. C. Rai, Nirupama Mallick,”Impact of aluminium, fluoride and fluoroaluminate complex on ATPase activity of Nostoc linckia and Chlorella vulgaris”,Biometals 9 (1996)

33 UPTAKE OF FLUOROALUMINATE SPECIES BY HUMANSLaboratory investigations have often used aluminofluoride complexes for stimulation of various guanine nucleotide binding proteins.These complexes form spontaneously in aqueous solutions containing fluoride and traces of aluminum and appear to act as phosphate analogous.In view of the ubiquity of phosphate in cell metabolism and together with the dramatic increase in the amount of reactive aluminum now found in ecosystems, aluminofluoride complexes represent a strong potential danger for living organisms including humans.Fluoride anions in the presence of aluminum affect the liver as an organ involved in glycogenolysis, fatty acid oxidation, and lipolysis.Enormous possibilities for multiple molecular interactions of aluminum and fluoride exist in the brain.

35 MY OPINIONFluoride or Fluoroaluminte complexes being directly responsible for CKDu is quite unlikely but blocking of glomeruli with precipitated CaF2 and proteins is possible.However, fluorosis prohibits the detoxification mechanism of bones.Therefore, patients with fluorosis are more prone to get their kidneys damaged by other toxic chemicals such as cadmium and arsenite ions.F- can inhibit various enzymes and HF can etch anything causing cancers.

36 CADMIUM ION HYPOTHESIS [25-27]Hypothesis of Cd2+ causing CKDu by Sarath Bandara et al. appears to the third such hypothesis.Elevated Cd2+ Levels in rice and Freshwater Fish.Both Rice and Inland Fish are a popular food among the farming community in this area.Cd2+ is a common poison present in TSP fertilizers.Atomic Absorption Spectroscopic determination of Cd2+ in rice and freshwater fish.Concentration of Cd2+ in roots of Nymphaea lotus L (Lotus roots)[25] J.M.R.S. Bandara et al. “Chronic Renal Failure among Farm Families in Cascade Irrigation Systems in Sri lanka Associated with Elevated Dietary Cadmium Levels in Rice and Freshwater Fish (Thilapia)”, Environmental Geochemistry and Health, 30 (2008)[26] J.M.R.S. Bandara et al., “Chronic Renal Failure in Sri Lanka caused by Elevated Cadmium Levels: Trojan Horse of the Green Revolution”, Toxicology Letters, 198 (2010a)[27] J.M.R.S. Bandara et al., “Pollution of River Mahaweli and Farmlands unde Irrigation by Cadmium from Agricultural Inputs Leading to a Chronic Renal Failure Among Farmers in NCP”, Environmental Geochemistry and Health, DOI /S

37 PROBLEMS OF THE ANALYTICAL TECHNIQUECalibration plot drawn in a concentration range higher than those in the analyte samples.Calibration plot is in the Linear Dynamic Range.Beer-Lambert Law: A = εlc, where A is the absorbance, ε the molar absorption coefficient of the sample, l the path length and c is the concentration of Cd2+.No guarantee for the holding of the law below or above the linear dynamic range.As such, the magnitudes of values may be different to actual values. This has been pointed out by Chandrajith et al. [28].USE OTHER TECHNIQUES SUCH AS ANODIC STRIPPING VOLTAMMETRY![28] Rohana Chandrajith, Shanika Nanayakkara, Kozuyoshi Itai, T.N.C. Aturaliya, C.B. Dissanayake, Thilak Abeysekara, Kouji Harada, Takao Watanabe and Akio Koishumi, “Chronic Kidney Diseases of Unknown Aeitiology in Sri Lanka, Geographical Distribution and Environmental Implications”, Environmental Geochemical Health, 33 (2011)

38 DEMONSTRATION OF THE PROBLEMSKIPDEMONSTRATION OF THE PROBLEMEXTRAPOLATION OF THE STRAIGHT LINE IS INCORRECT PARTICULARLY IN THE HGH CONCENTRATION SIDE. IN THIS CASE THE TREND IN THE LOW CONCENTRATION SIDE MAY BE CORRECT SINCE THE LINE GOES TO THE ORIGIN. THIS IS NOT THE CASE ALWAYS!

39 BIOACCUMULATION OF CADMIUM IONS SKIPAlthough Cd2+ levels in surface water may be lesser than what Bandara et al. have reported, Cd2+ has a notorious bioaccumulation and a very long biological half life (vide infra).Therefore, inland fish may have high concentrations of continually accumulated Cd2+.Once Cd2+ is ingested continuously, they remain in the human body for a long time causing chronic problems.

40 CADMIUM IN FISH Measured in flesh extract.Cd2+ concentrates in the bones by exchanging for Ca2+ ions.In this way, Cd2+ accumulates in fish bones.Ca10(PO4)6(OH)2(s) + yCd2+(blood) ⇌ Ca10-yCdy(PO4)6(OH)2(s) yCa2+(blood)Cd2+ may be found in liver and kidneys as well. Mechanisms will be discussed later.

41 CADMIUM POISONINGCadmium toxicity occurs when a person breathes in high levels of cadmium from the air, or eats food or drinks water containing high levels of cadmium. Cadmium is found inBatteries: Ni/Cd Batteries: Major Cadmium Source.PigmentsMetal coatingsPlasticsSome metal alloysFertilizers: IMPORTED TSP: [Cd2+] = 71.7 mg Cd2+/kg P2O5 [29].Since 1973 to 1984, 68.9 tonnes of Cd has been added to agricultural lands. EPPAWELA PHOSPHATE HAS VERY LOW Cd2+ CONTAMINATION!CigarettesStabilizer in PVC

42 RISK FACTORS SKIP Smoking.Living near hazardous waste sites or industrial factories that emit cadmium into the air.Working in a metal smelting and/or refining plant.Working in a plant that produces cadmium products (e.g., batteries, coatings, plastics, pigments).Having a nutritional deficiency in calcium, iron, protein, and/or zinc [THESE PEOPLE USUALLY DO NOT TAKE A HEALTHY DIET].

43 SYMPTOMS OF CADMIUM POISONING SKIPEating food or drinking water contaminated with high levels of cadmium can result in: Vomiting/nausea, Stomach cramps, Diarrhea, Kidney damage, Fragile bones and Death. SYMPTOMS OF THE ITAI-ITAI DISEASE Breathing in cadmium can result in: Lung damage (chest pain or shortness of breath), Kidney diseases, Fragile bones and Death.

44 DIAGNOSIS (SKIP DUE TO TIME LIMITATIONS)Your doctor will ask about your symptoms and medical history, and perform a physical exam.Tests may include the following:Blood testsUrine testsHair or nail analysisNeutron activation analysis—a test to measure cadmium levels inside your liver and kidneys.

45 TREATMENT (SKIP DUE TO TIME LIMITATIONS)There is no effective treatment for cadmium toxicity.Avoid exposure.Your treatment will be designed to help manage and relieve your symptoms.You may be given vitamin D for the weak bones.

46 To help reduce your chances of getting cadmium toxicity, take the following steps: SKIPDo not smoke. Smoking is the single most important source of cadmium intake for most persons.Identify potential sources of cadmium in and around your home, at work, and where your children play.If you maintain a vegetable garden, consider having fertilizers tested for cadmium. Some fertilizers have been found to be high in cadmium, which may then concentrate in your vegetables. Avoid any use of cadmium containing fungicides near your vegetable gardens.Eat a balanced diet that provides enough calcium, iron, protein, and zinc.Take inventory of and properly store (out of the reach of children) cadmium-containing products in your home (e.g., fungicides, batteries, metals, fabric dyes, ceramic/glass glazes, fertilizer); check the label for cadmium.

47 Keep nickel-cadmium batteries out of the reach of small children and find out how to properly dispose of these batteries.Read instructions for safely using cadmium-containing fungicides or fertilizers on your lawn or garden.If you have a water well, have your water tested for the presence of cadmium.If cadmium is present in your well water, consider using bottled water for drinking or install a water filter that removes cadmium and other metals from drinking water.Do not allow young children to play in or around hazardous waste sites.SKIP

48 ITAI-ITAI DISEASE: DUE TO CADMIUM POISONINGItai-tai disease is caused by cadmium poisoning due to mining in Toyama Prefecture of Japan. Cd2+ goes in through rice and cereals!This is a multi-factorial disease but cadmium is a necessary agent to result in severe kidney damage and osteomalacia (Osteomalacia is derived from Greek: osteo- which means "bone", and malacia which means "softness". ).

49 can coordinate with Cd2+.LIGNINLatifoliate: Plants with broad leaves.Cadmium ions have a high affinity to rice and cereals. Recent discovery of Cd2+ accumulation in red onions is also a sensible finding.Allyl propyl disulfidecan coordinate with Cd2+.Nelum roots have glucose, fructose, sucrose, mannitol, galacturonic acid, raffinose, 10 amino acids and fibers [29]. Cadmium ions can form coordination complexes with them.[29] S. Hujjatullah, A. K. Bloch and A. Jabbar, ” Chemical composition and utilisation of the roots of Nymphaea lotus L”, Journal of the Science of Food and Agriculture, 18 (1967)

50 Cadmium and the Kidney [30] SKIPChronic cadmium exposed battery workers had major signs in kidneys: Tubular and Glomerular dysfunction with proteinuria.A high prevalence of kidney stones in Swedish and British Workers.Glomerular dysfunction: Damaging glomeruli and letting red blood cells leak into urine.Proteinuria: Presence of an excess of serum proteins in urine[30] L. Friberg, “Cadmium and the Kidney”, Environmental Health Perspectives, 54 (1984) 1-11.History and Aetiology of Cadmium-induced Kidney Dysfunction and Related Disorders

51 Cadmium Metabolism, Metallothionein and Kidney DysfunctionAfter the ingestion, cadmium is transported to the liver where it stimulates the synthesis of metallothionein.Cadmium is then bound to metallothionein and transported via blood to kidneys.L-CysteineMetallothionein (MT) is a family of cysteine-rich, low molecular weight (MW ranging from 500 to 14,000 Da) proteins. In the human body, large quantities are synthesized primarily in the liver and kidneys.

52 KIDNEY DAMAGE BY CADMIUMCadmium bound to metallothioneins are filtered with the primary urine and reabsorbed into the tubular cells.A considerable catabolism of cadmium-metallothionein can take place.Complexes break and cadmium ions bind to newly formed metallothionein in the tubular cells.Kidney damage takes place when the kidney no longer produces metallothionein.The biological half-life of cadmium is very long: Years!Chronic cadmium exposures of 50 µg m-3 (10 times the new OSHA permissible exposure limit) for 10 years may cause kidney dysfunction with proximal tubular damage [30].[30] Human Toxic Chemical Exposure-Cadmium, “Bulletin of Pacific Toxicology Laboratories”,

53 RENAL TOXICITY OF CADMIUM IONSThe kidney is the major target organ of cadmium.The critical concentration for toxic effects appears to be between 200 and 250 µg/g of kidney tissue ( ppm) [31]When the renal tubular cells become saturated with cadmium (because the storage capacity of metallothionein is exceeded) free cadmium is released.It is widely believed that this free cadmium ion is the cause of the renal damage.Free cadmium ions may replace zinc in some systems, especially those enzymes involved in the reabsorption and catabolism of proteins.[31] Roels, H., Bernard, J., Goret, A., Lauwerys, R. Chettle, D., Harvey, T., Al Haddad, I., "Critical concentration of cadmium in renal cortex and urine" Lancet i, 221, 1979.

56 SUBSTITUTION OF Cd2+ FOR Zn2+ IN Zn2+-CONTAINING ENZYMESIn a variety of metalloenzymes, the active site contains a catalytically-essential, tetra-coordinated Zn2+ ions.Three of the ligands to the metal ion are provided by amino acid side chains, generally in distorted geometrical relationships, while a water molecule serves as the fourth ligand.Chief examples of such enzymes are carboxypeptidase A, thermolysin, carbonic anhydrase, alcohol dehydrogenase and alkaline phosphatase.

57 The male farmers usually drink excessive amounts of illicit alcoholThe male farmers usually drink excessive amounts of illicit alcohol! The water used to dilute spirit may contain anything. Hint for male patients? They work hard under hot sun.Dehydration results in concentration of species present in blood. Alcohol consumption further aggravates dehydration.Cadmium ions, if ingested, can replace the Zn2+ in alcohol dehydrogenase and thereby can affect detoxification mechanism of toxic ethanol from the liver.Cadmium ions bound to alcohol dehydrogenase can go to kidneys and damage them.Alcohol taking farmers who have got Cd2+ through food and/or drinking water are more susceptible to several illnesses including kidney damage.Besides, male farmers are the people who spray toxic chemicals to paddy fields without having protective clothing and face masks.

58 ANALYSIS OF Cd2+-INDUCED RENAL TUBULAR DAMAGESeveral proteins including retinol binding protein and NAG have been suggested as markers for proteinuria, but ß-2-microglobulin in urine appears to be the most widely used and best characterized analyte to evaluate cadmium-induced renal tubular damage [35]. [35] U.S. Department of Labor OSHA, 29 CFR Parts 1910, 1915, 1920 "Occupational Exposure to Cadmium; Final Rules" Federal Register vol. 57, no. 178, Monday, September 14, 1992, Rules and Regulations.

59 FOURTH HYPOTHESIS-CYANOBACTERIABy Dhammika M. Dissanayake et al.SKIP DESCRIPTIPONCyanobacteria also known as Cyanophyta, is aphylum of bacteria that obtain their energy through photosynthesis.They release O2 to the atmosphere an hence they have changed the early reducing atmosphere into oxidizing atmosphere for other biological species to evolve.

60 FACTS ABOUT CYANOBACTERIA: SKIPAquatic cyanobacteria are known for their extensive and highly visible blooms that can form in both freshwater and marine environments.The blooms can have the appearance of blue-green paint or scum.These blooms can be toxic, and frequently lead to the closure of recreational waters when spotted. Marine bacteriophages are significant parasites of unicellular marine cyanobacteria.

61 WHAT CYANOBACTERIA BLOOMS LOOK LIKE: SKIPLook like foams, scum or mats of the surface of fresh water lakes and ponds.The blooms are blue, bright green, brown or red.They may look like paint floating on water.As algae in cyanobacterial blooms die, the water may smell bad.

62 CYANOTOXINS SKIPSome cyanobacteria produce toxins, called cyanotoxins.Among cyanotoxins are some of the most powerful natural poisons known, including poisons which can cause rapid death by respiratory failure. The toxins include potent neurotoxins,hepatotoxins, cytotoxins, and endotoxins.

64 FURTHER STUDIES ON CYANOBACTERIAL TOXICITY TO KIDNEYS SKIPSome results suggest that kidney impairment from chronic exposure of toxic cyanobacterial blooms might be the first step, and then followed by hepatic failure [37]. Studies are also underway at the Institute of Fundamental Studies. Group Leader is D. N. Magana-Arachchi.[37] Qiu T, Xie P, Li L, Guo L, Zhang D, Zhou Q, “Nephrotoxic effects from chronic toxic cyanobacterial blooms in fishes with different trophic levels in a large Chinese lake.”, Environmental Toxicological Pharmacology, 33 (2012)

65 THE CYANOBACTERIAL TOXINS: A HIDDEN HEALTH HAZARD: By Dhammika MTHE CYANOBACTERIAL TOXINS: A HIDDEN HEALTH HAZARD: By Dhammika M. Dissanayake [38]According to their ongoing research studies, it is substantiated that toxins produced by the cyanobacteria may have a role in the pathogenesis of CKDu in Sri Lanka.Further, they have found that the cyanobacterial toxins could be the causative agents for the nephropathy in Balkan countries because there are very close similarities between Balkan Nephropathy and CKDu in Sri Lanka.Environmental factors such as global warming and admixing of Nitrogen, Phosphate and Potassium of chemical fertilizers with run off water provide a favorable ecological background for the blooming and toxin production of cyanobacteria.The management of patients with renal disease is expensive and need long term care.This is an unbearable burden to the economy of the country.As such , short-term and long -term preventive strategies like introduction of new water sources, measures to control the usage of chemical fertilizers and control of global warming are mandatory to alleviate the disease in addition to early detection.THIS IS A GOLDEN SUGGESTION!!!!

66 SKIP BALTIC CDK [39] CYANOBACTERIA WORLD MAPFluoride in Drinking Water is Less.Fluoride is added to salt .Fluoride is added to drinking water.Balken Nephropathy affects only certain endemic rural foci along tributaries of the Dangue River in Balken Countries of Bosnia, Bulgaria, Croatia, Rumania and Serbia [39].IS IT GENETIC DISEASE THEN?[39] G. Bomias and J. Boletis, “Balken Nephropsthy, Evolution of Our Knowledge, American Journal of Kidney Disease, 52 (2008)CYANOBACTERIA WORLD MAP

67 OBJECTIONS FOR CYANOBACTERIA AND SRI LANKAN CKDuApparently, these farmers drink dug well (92%) and tube well (8%) waters [40].The cyanobacteria levels in these waters are marginal [41].[40] Priyani paranagama, “Potential Link between Ground Water Hardness, Arsenic and Prevalence of CKDu”, [Not a standard peer reviewed journal reference.][41] CKDu and Arsenic by the Arsenic Group [Not a Standard Journal Article]COULD NOT FIND RELIABLE STANDARD JOURNAL ARTICLES!PLEASE SEE LATER: THIS DUG AND TUBE WELL WATERS CAN NOT HAVE GLYPHOSATE ALSO. THIS GOES AGAINST THEIR GLYPHOSATE HYPOTHESIS.

69 LITTLE BIT OF ARSENIC CHEMISTRYCompounds of Arsenic exists in four Oxidation States; +5, +3, 0 and -3.As is not a true metal. It is a metalloid.In water, +3 and +5 Oxidation States DO NOT EXIST as As3+ and As5+ bare cations [That is why we write them as As(III) and As(V)] but they exist as oxyanions: arsenite (AsO33- or AsO2-) and arsenate (AsO43-).As such, Arsenic does not form coordination complexes as other cations do since As does not exist as a cation.Arsenes can act as ligands and can donate electrons to metal ions.Compounds of Arsenic are Covalent rather than Ionic.Arsenite salts are more soluble in water than arsenate salts.Solubility of Ca3(AsO4)2 in water is 0.13 g L-1 at 25 °C.Ca3(AsO4)2 is soluble in acids. If ingested, can dissolve in the liver.Ca3(AsO3)2 is soluble in water.

70 CKDu AND ARSENIC This group says:CKDu is NOT associated with known risk factors of CKD such as diabetes, hypertension and glorulonephritis.Clinically, the disease is associated with β-2-microglobulinuria.High levels of β-2-microglobulinuria is a feature of arsenic induced nephrotoxicity [43]. PLEASE SEE THE REFERENCE PROVIDED![43] F. Homg, T. Jin, A. Zhan, “Risk Assessment on Renal Dysfunction caused by Co-exposure to Arsenic and Cadmium using Benchmark Calculation in Chinese Population”, BioMetals ,17 (2004)All other articles in the same journal, same volume, are about β-2-microglobulinuria as a feature of Cd2+-induced nephrotoxicity [44].[44] George Kazantzis, “Cadmium, Osteoporosis and calcium metabolism”, BioMetals, 17 (2004)Several other papers are about Cd2+ poisoning, kidney dysfunction and β-2-microglobulinuria . We have described this previously [35]!

74 DERMATALOGISTS VIEWFROM THE RESULTS OF CKDu PATIENTS DIAGNOSED, THERE ARE NO EVIDENCE TO INDICATE THE PRESENCE OF PIGMENTATION AND KERATOSIS ON THE SKIN. NO EVIDENCE FOR MEES’ LINES THAT ARE COMMON TO PERSONS SUFFERING FROM ARSENIC TOXICITY [49].[49] Noble, A.; Amerasinghe, P.; Manthrithilake, H.; Arasalingam, S Review of literature on chronic kidney disease of unknown etiology (CKDu) in Sri Lanka. Colombo, Sri Lanka: International Water Management Institute (IWMI). 41p. (IWMI Working Paper 158). doi: /MEES’ LINES

75 MECHANISM OF CELL DEATH PROPOSED BY THE ARSENIC GROUPBinding of Ca3(AsO4)2 to arsenic transporters in the liver and transport to the kidneys. What are these transporters? Metallothinine is to transport Zn2+ but Cd2+ interferes strongly. Arsenate or arsenite can not bind to metallothionines.Deposition of Ca3(AsO4)2 crystals in the kidney tissues.Release of arsenate groups. This is an ionic compound. Release should depend on water solubility. This compound is NOT water soluble.Replacement of Phosphate Groups by Arsenate Groups and DNA Damage.THESE ARE ONLY SPECULATIONS WITHOUT ADEQUATE DATA OR LITERATURE SUPPORT! SOME ARE THERMODYNAMIC IMPOSSIBILITIES!Replacement of PO43- by AsO43- in DNA is thermodynamically forbidden and hence can not take place. DNA does not contain arsenic as an element even in “Arsenic Loving Bacteria” found in the California lake.

76 SKIPChronic inhalation exposure to elevated levels of inorganic arsenic in humans is associated with irritation of the skin and mucous membranes (dermatitis, conjunctivitis, pharyngitis, and rhinitis).Chronic oral exposure to elevated levels of inorganic arsenic in humans has resulted in gastrointestinal effects, anemia, peripheral neuropathy, skin lesions, hyperpigmentation, gangrene of the extremities, vascular lesions, and liver or kidney damage.Some recent studies have reported an association between elevated arsenic levels in drinking water and neurocognitive or behavioral test results in school age children.

77 SKIPIngestion of inorganic arsenic in humans has been associated with an increased risk of nonmelanoma skin cancer and also to an increased risk of bladder, liver and lung cancer.The Reference Dose (RfD) for inorganic arsenic is mg/kg/day (i.e ppm per day, very low, it is such a bad poison!) based on hyperpigmentation, keratosis, and possible vascular complications in humans. The RfD is an estimate (with uncertainty spanning perhaps an order of magnitude) of a daily oral exposure to the human population (including sensitive subgroups) that is likely to be without appreciable risk of deleterious noncancerous effects during a lifetime.

78 SKIPArsenic-containing pesticides may still be found in some U.S. farms and homes [ATSDR 2007].Gallium arsenide is used in integral components of discrete microwave devices, lasers, light-emitting diodes, photoelectric chemical cells, and semiconductor devices.Other industrial processes that use arsenic include coal-fired power plants, hardening metal alloys, and purifying industrial gases (removal of sulfur).Inorganic arsenic is found in industry, in copper chromated arsenate treated lumber, and in private well water in some parts of the USA.

79 Seafood (especially bivalves [clams, oysters, scallops, mussels], crustaceans [crabs, lobsters], and certain cold water and bottom feeding finfish, and seaweed/kelp.The organic forms of arsenic found in seafood (mainly arsenobetaine and arsenocholine, also referred to as “fish arsenic”) are generally considered to be nontoxic, and are excreted in urine within 48 hours of ingestion [ATSDR 2007].However, inorganic forms of arsenic have been found in some types of seaweed.Recent literature suggests hijiki seaweed has very high levels of inorganic arsenic (MMA) [Rose et al. 2007].ARSENIC IN FOODSKIP

80 Lead Arsenate PesticidesSKIPLead Arsenate PesticidesBoth lead and arsenic can be toxic at high concentrations in soils.Lead: Preventing lead exposure in infants and young children is important because lead can affect their developing brain and nervous system. High levels of lead can adversely affect the nervous system and kidneys of adults and children.Lead hydrogen arsenate, also called lead arsenate, acid lead arsenate or LA, chemical formula PbHAsO4, is an inorganic insecticide used primarily against the potato beetle.

82 INORGANIC ARSENIC SPECIS ARE HIGHLY TOXICSKIPThere is much debate on Napoleon's death, whether he was poisoned by arsenic-tainted wine during his exile on the Island of St. Helena.More than 20 Arsenic Species are present in the Natural Environment and Biological Systems.Although the trivalent arsenic species, such as inorganic arsenite (AsIII), monomethylarsonous acid (MMAIII), and dimethylarsinous acid (DMAIII) are highly toxic, arsenobetaine (AsB), the predominant arsenic species present in most crustaceans, is essentially non-toxic [51].[51] X. Chris Lee, “Arsenic Exposure, Metabolism and Health Effects”, Home Page: Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta

83 ARSENIC METABOLISM [52] SKIPBiomethylation was thought to be a detoxification mechanism of arsenic species. However, these organoarsene compounds are soluble in fat and in blood serum.Biomethylation in the liver is the main mechanism for the metabolism of inorganic arsenic. It involves alternate steps of two-electron reduction followed by oxidative addition of a methyl group.[ 52]

85 GLYPHOSATE AND CKDuGlyphosate (N-(phosphonomethyl)glycine) is a broad-spectrum systemic herbicide used to kill weeds, especially annual broadleaf weeds and grasses known to compete with commercial crops grown around the globe.It was discovered to be a herbicide by Monsanto chemist John E. Franz in SKIPMonsanto brought it to market in the 1970s under the trade name "Roundup", and Monsanto's last commercially relevant United States patent expired in 2000.In alkaline conditions this exists as an anion.

86 GLYPHOSATE ACTION SKIPGlyphosate kills plants by interfering with the synthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan.It does this by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthatase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (ESP) [55].[55] H.C. Steinrücken, N. Amrhein , "The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase“, Biochemistry and Biophysics Research Communications, 94 (1980) 1207–1212.[56] Image :

88 ENVIRONMENTAL FATE OF GLYPHOSATE [59 ]Glyphosate adsorbs to soil down to 6 inches of soil layer.Readily degraded by microbes to aminomethylphosphonic acid and carbon dioxide.Glyphosate and residues can not contaminate ground water because they do not move that down due to adsorption by surface soil. The Arsenic Group says that these patients drink/drank well waters {92% Dug Wells and 8% Tube Wells!}.Can contaminate with surface waters.The half-life of glyphosate in soil ranges between 2 and 197 days; a typical field half-life of 47 days has been suggested.The median half-life of glyphosate in water varies from a few days to 91 days.[ 59] National Pesticide Information Center

91 GLYPHOSATE AND ARSENICTHERE IS AN OPEN ACESS JOURNAL PAPER.[67] Channa Jayasumana, Sarath Gunathilaka and Priyantha Senanayake, “Glyphosate, Hard Water and Nephrotoxic Metals: Are They the Culprits Behind the Epidemic of Chronic Kidney Disease of Sri Lanka?”, International Journal of Environmental Research and Public Health, 11 (2014)This Glyphosate scandal came out due to THIS paper [67]!

92 They talk about glyphosate-divalent and trivalent metal complexesThey talk about glyphosate-divalent and trivalent metal complexes. The reference given is a DFT Computer Simulation Study. Only theoretical possibilities [68].IMPORTANT POINT IS THAT “None of these complexes have arsenic even as arsenate. I have the paper. You know that arsenic does not form complexes by behaving as a metal ion. Replacement of phosphate by arsenate is not possible either![68] Caetano, M.; Ramalho, T.; Botrel, D.; da Cunha, E.; de Mello, W., “Understanding the inactivation process of organophosphorus herbicides: A DFT study of glyphosate metallic complexes with Zn2+, Ca2+, Mg2+, Cu2+, Co3+, Fe3+, Cr3+, and Al3+.”, International Journal of Quantum Chemistry, 112 (2012) 2752–2762.

93 I have requested Dr. Udayanga Ranatunga to do the DFT study on possible complexes of metal ions, arsenic and glyphosate.Even if theoretically predicted, that does not mean that the compounds are practically synthesizable.

94 SKIPOther reference quoted for glyphosate-arsenic polymeric complex isTHIS IS A BUSINESS REPORT ON ORGANIC DOG FOODS. AN ADVERTISEMENT!The polymeric complex in [67] is not contained in this pdf as well!

95 AS A CHEMIST’s POINT OF VIEW!At high pH, Glyphosate exists as anion with carboxylate and phosphate groups. Arsenate and Arsenite are also an anions. Naturally, they have no affinity to each other.In the high acidic environment of the liver, glyphosate carboxyl group and the phosphate group can get protonated. Arsenate or arsenite also get protonated.Some hydrogen bonding between them are possible in the liver.However, blood has almost neutral pH [pH = 7.4, tightly regulated between 7.35 and 7.45]. These hydrogen bonds are not stable then.So, I DO NOT understand how glyphosate is carryings arsenic species from liver to kidneys.Glyphosate can carry Cd2+ by forming coordination complexes with metallothionines.However, glyphosate can make better coordination complexes with other metal ions such as Zn2+, Mn2+, Co3+, Fe(II) and Fe(III) which are present in relatively higher concentrations when compared to that of Cd2+ in human body.Therefore, glyphosate can induce essential metal ion deficiencies. It can be toxic.

97 BETTER AVOID SYNTHETIC FERTILIZERS AND AGROCHEMICALS, ANYWAY!Whether they contribute to CKDu or not, they are poisons.It is indeed better to avoid them, if possible.Organic farming is good but the yields are less. Better to cultivate increased amounts.Cost of organically grown food stuffs are high though there is no certificate that these were grown without agrochemicals.Weed picking by hands as the way it was done in the past is NOT practical now.Feeding 21 million population in this country is also a prime concern or they will die due to starvation.If possible, better to grow vegetables and fruits in your own garden!

98 RENAL DIALYSIS A Patient Receiving Dialysis A process for removing waste and excess water from the blood.Used as an artificial replacement for lost kidney function in people with renal failure.The kidneys have important roles in maintaining health.When healthy, the kidneys maintain the body's internal equilibrium of water and minerals (sodium, potassium, chloride, calcium, phosphorus, magnesium, sulfate). The kidneys also function as a part of the endocrine system, producing erythropoietin and calcitriol. Erythropoietin is involved in the production of red blood cells and calcitriol plays a role in bone formation.Dialysis is an imperfect treatment to replace kidney function because it does not correct the compromised endocrine functions of the kidney.Diffusion through concentration gradient and ultrafiltration are the principles involved. Semi -permeable membrane with different pore sizes is used.

99 ROLE OF NANOTECHNOLOGY IN RENAL SUBSTITUTION THERAPYTwo Membranes called G Membrane and T Membrane.G membrane resembles the function of the glomerular basement membrane.G Membrane is a fully porous membrane which removes solute up to the molecular weight of albumin. Ultra-filtrate passing through the G Membrane passes through the T Membrane.T Membrane is meant to emulate the tubular membrane in the renal tubules. T membrane will reabsorb all of those substances we want to retain, some sodium, some potassium, calcium, a little bit of phosphorous, and so on. T Membrane is Nano-technologically Developed Membrane with very small pores ~ 1 nm length and the distance between pores is 1 – 5 nm.Dean G. Johnson , Tejas S. Khire, Yekaterina L. Lyubarskaya, Karl J.P. Smith, Jon-Paul S. DesOrmeaux, Jeremy G. Taylor, Thomas R. Gaborski, Alexander A. Shestopalov, Christopher C. Striemer, James L. McGrath, ‘Ultrathin Silicon Membranes for Wearable Dialysis”, Advances in Chronic Kidney Disease, 20 (2013)

105 STEM CELL-BASED KIDNEY REGENERATION[74] S. Yokote and T. Yokoo, “Stem cells in kidney regeneration”, Current Medicinal Chemistry, 19 (2012)[75] Brian A. Yeagy & Stephanie Cherqui , “Kidney repair and stem cells: a complex and controversial process”, Review, Pediatric NephrologyDOI /s x, Published Online 19 February, 2011.Kidney grown from stem cells by Australian scientistsAustralian scientists grow world's first kidney from stem cells in a breakthrough that could alleviate the demand for organ transplants, Journal Nature Cell Biology,"This is the first time anybody has managed to direct stem cells into the functional units of a kidney," Professor Brandon Wainwright, from the University of Queensland, told The Telegraph.[76] Kidney disease: how could stem cells help? Last updated: 12 Aug 2013AND 100s MORE ARTICLES!THIS MIGHT BE THE WAY OUT FOR TREATING POOR PATIENTS!

106 SUMMARY CKDu is a serious epidemic in Sri Lanka.F- Hypothesis: HF can damage liver. Fluorosis prohibits bone’s detoxification mechanism. F- can cause kidney damage.Fluoroaluminate complexes are toxic. Reports on Renal Failure are available.Cd2+ is present in TSP fertilizers and in some weed killers. Cd2+ is a well known nephrotoxic ion. Cd2+ is present in cigarette smokes.Arsenic species are poisons. Speculations are not scientific research.Metal ion-arsenate-glyphosate polymers have not been synthesized.No evidence to suggest connections between arsenic species and glyphosate being responsible for CKDu.This does not mean that they are not toxic.It is very likely that increased concentrations of all the ions in the blood is responsible for the denaturing of glomeruli proteins. Ions at the top of Hofmeister series are more dangerous than other ions.Research on Implantable Kidneys is important.Regenerative Medicine may be a possible way to treat CKDu Patients. However, it is still at the research level.